August 15, 2010OA & Vt group projectqPCR of cleaned RNA from 8.14. Using cjun kinase primers, PCR of 8.7 A & B and 8.8 G & H to test for genomic DNA contamination.

9:30 am: spawned oysters. 4 females and 4 males. Pooled all sperm and all eggs. Had 3 flasks/bottles for each CO2 (~400 ppm and ~900 ppm) treatment. Divided eggs between 6 vessels - 20 mL of eggs per vessel. Put in 2 mL of sperm to each vessel. Tracked development in each vessel taking pictures and video with the microscope (see schedule in 8.14.10).

New experiment: OA effects on fertilization and development (OA Dev.). Will fertilize eggs in differing CO2 conditions and track major development milestones. If larvae are still alive at 48 hours, will dose with Vt. See below for detailed timeline.Collected 9 more adult oysters (C. gigas) from Argyle Creek. Put in sea tables overnight.

EF1 amplified the same gene product and was ~the same for both pooled groups. All other successful primers amplified identical gene products and showed potential for differential expression. The only 2 primers that didn't work were BPI and SOD. qPCR for 9 pm with primers EF1, Prx6, C-jun, and chit protein.

Lisa and I both counted larvae per mL in large bucket of larvae. We each counted 1 mL of larvae in triplicate. Counts for Emma: 50, 79, 105. Counts for Lisa: 61, 82, 94. Determined we have about 80 larvae per mL. Larvae look like trocophores with small velum.
Measured Moose's pCO2 & pH4 pm (48 h post-fertilization): Divided larvae equally between 8 treatment chambers. 4 chambers with larvae were at ~400 ppm and the other 4 were at ~800 ppm CO2. Took 2 samples from bucket in RNAlater for baseline.

August 12, 2010OA & Vt group project9 am: larvae in buckets observed actively swimming under scope and are in trocophore stage.~10 am: Pulled off upper layer of water in both buckets and combined in ~10 L filtered seawater. Debris/unfertilized eggs on bottom discarded. Larvae fed 50 mL of T. iso and aerated.

Measured pCO2 of OA system and pH + temperature of Moose's limpet experiment.

Reverse transcribed RNA extracted from larvae 8.5.10 and 8.6.10 (see protocol November 6, 2009 in Roberts lab notebook). Diluted cDNA with 225 uL water. Pooled 4 uL of I & B from both days to make 16 uL of CC (control CO2, no Vt) for pooled q PCR; did the same for G & H to be pool of CO2V cDNA. Made primer dilutions (10 uM) for qPCR tomorrow: BPI, EF1, cjunk, SOD, Prx6.

Determined sex of each oyster by smearing some of the gonad on a slide. Sperm were tiny and very mobile; eggs were larger, somewhat rectangular-shaped and had a translucent circle in the center. Eggs were kept separately and sperm were combined in one jar. In total, 4 females and 3 males were spawned. The mixed sperm from the 3 males was added gradually to each of the 4 female jars (all in filtered seawater), ~1-2 mL at a time (3 times). It was added gradually to avoid complications with polyspermy. Still, too many sperm were added - under scope could see dozens of sperm clustered around each egg. Ever 1-2 hours for the first 6 hours let eggs settle and poured off upper layer of liquid, replacing with filtered seawater.

About 6 hours post-fertilization cleavage was visible and oocytes were divided between 2 5-gallon buckets, each filled with ~6 L of filtered seawater. Buckets were aerated with air pumps and left to sit on the benchtop until morning.

August 9, 2010OA & Vt group projectSampled larvae for mortality estimates and RNA analysis. Went back to previous method of mortality sampling, which is plunging chambers before filtering and sampling and then later sampling for RNA analysis. No CO2, pH or temperature data for water supply today since probes are broken. Temp in set-up was 19C (as it has been every day).

Extracted RNA from larval samples taken 8.5 and 8.6 (n=8 from each day) using Tri Reagent (manufacturer's protocol). Samples were resolubilized in 50 uL DEPC-treated H2O and stored at -80C.

August 7, 2010OA & Vt group projectColonies of Vt did not grow on new plates.

Sampled larvae for mortality estimates and RNA analysis.

August 6, 2010OA & Vt group projectPlates of Vt were contaminated and uncountable. Replated serial dilutions of the same culture (now 48 hours) in lab 10 using new plating materials and plates from Sammi. Serial dilutions were made in DEPC-treated water.

Sampled larvae for mortality estimates and RNA analysis. Changed sampling for mortality - now sample larvae after have filtered all out and sampled for RNA.

Extracted the last 18 samples from week of July 12. 4 samples from yesterday were left out overnight: H 7.16.10; B 7.14; D 7.15; F 7.14.

August 5, 2010OA & Vt group projectTried to spec Vt grown yesterday at 620 nm but only got an OD of 0.14, which corresponded to about 0 CFU/mL (obvious Vt growth in tubes and not in control). Plated serial dilutions of 100 uL for 10^-3 through 10^-8 on marine agar. Incubated 24 hours in lab 10. Put 1 mL of broth + Vt into each of 3 microfuge tubes and spun down at max speed for 3 minutes. Removed supernatant and resuspended in 1 mL of sterile seawater.morning: Changed water and sampled larvae from all chambers for RNA and for mortality (mortality estimates in xls). Inoculated 2 chambers in each treatment with 30 uL resuspended Vt. Initially all 4 chambers in the control CO2 were inoculated so had to switch 2 of the inoculated controls with 2 uninoculated high CO2. Will take pH readings in the afternoon to see if bubbled in gas equilibrated water to correct treatment. The controls switched in to the high CO2 were G and H; the high CO2 switched in to the controls were I & B. A,L, G and H are inoculated. Controls of seawater for just Vt were also inoculated.

Extracted RNA from 24 more T/Vt larval samples.

August 4, 2010OA & Vt group projectmorning: Began experiment. Put Cg larvae in 8 different chambers in 19C circulating water bath. System is static. Added water at ~380 ppm (control) to 4 of the chambers and at ~900 ppm (treatment) to the other 4. All chambers have ambient (control) or treatment CO2 bubbling in. See data spreadsheet for detailed information on treatment CO2, pH, T and salinity.
Inoculated 2 50-mL falcon tubes with Vt grown on agar 8.1.10. Also made 1 50mL control. Incubated on shaker in lab 10 for 24 hours.

Extracted RNA from 24 Cg larval samples taken during the temperature/Vt trial week of July 12. Used Tri Reagent and followed manufacturer's protocol. Reconstituted RNA in 50 uL DEPC-treated H2O and stored at -80C.

155*(10^6)*10

1.55 * 10^9 CFU/mLChambers were inoculated at 10^4, 10^6 and 10^8 Vt.

CF did a spot check of the control larvae and saw some swimming. in general concern about amount of marine broth in control and treatment chambers because water was murky and smelled funky in the 10^-1 concentrations. Especially in the vibrio 10^-1 there were very few larvae in the water column, most were stuck around the stopper at the bottom. The algae particles had also sunk and were stuck to the outside of the chambers, causing fouling. Wiped out all algae before refilling chambers. Did not re-inoculate. Fed all chambers 25 mL algae mix.
Neutral red did not work - saw some clear larvae that were obviously alive. ~4:30 pm took 10 mL of water from each chamber. Under dissecting scope, counted live larvae as observed by swimming, live larvae as observed by ciliary action, and dead larvae ( I counted larvae from the Vt 10^-5 chambers, Loni counted all others). % mortality graph below; values are in a google spreadsheet. Due to bias in counting depending on person determined that there is probably no significant mortality at this stage.

Serum Agglutination Test
See protocol . In 2 well depression slide made control and test agglutination mixes. Control was 50 uL sterile seawater + 25 uL Vt in marine broth. Test was 25 uL SSW, 25 uL Vt in broth and 25 uL polyclonal antibody (made in Ralph's chicken). Both wells were mixed gently with pipette (separate tips) and left to incubate for 5 minutes. Under 10x, obvious clumping for test well and free-floating bacteria in control (starry sky vs. glaciers).
Control

August 1, 2010OA & Vt class project
Changed the water in all larval chambers and took samples for mortality estimates and ~1000 larvae in 0.5 mL RNAlater for gene expression analysis. For mortality, larvae were put in a 6-well plate in 5 mL of seawater + 5 uL 1% neutral red and left to incubate for 6 hours. Live & dead were counted at the end of 6 hours for each chamber. Samples in RNAlater were stored at 4 C.
No bacteria grew in the control culture and the inoculated cultures were visibly cloudy. Plated 100 uL of culture dilutions 10^-3 through 10^-8 (done by Morgan) in duplicate. Plates were incubated at RT in Lab 10 and cultures were put back on shaker.
Larval chambers were inoculated as follows: E & L were controls and inoculated with 10^-1 concentration of marine broth (150 mL). G & H were inoc. with 10^-1 of Vt culture (150 mL), I & B with 10^-3 Vt (1.5 mL), and A & P with 10^-5 Vt (15 uL).
All chambers were fed 25 mL algae mix.
~1/2 of the water in the 5 gallon bucket was filtered out and replaced with fresh water. Larvae were given more algae.

July 31, 2010Protein gel & Western Blot
WB redone with barnacle protein extractions (done by Loni & Marie) worked with antibody for heat-shock protein. Conclusion that antibody does not work well with other marine inverts.

OA & Vt class project
Changed the water in all larval chambers and took samples from each for microscopy. Larvae were mostly swimming actively and appeared healthy. Fed all larvae 25 mL of algae mix.
Morgan and Kathy worked on the Vt cultures. They made 3 L of marine broth, 1 for control (no Vt) and two to inoculate with Vt from a plate culture. Flasks were all incubated in Lab 10, the 2 cultures were put on the shaker.

July 30, 2010Plated hemocytes (O. edulis)
There were things growing on the plate, most likely bacterial colonies. Plans for further experiment: incubate hemocytes alone and +V. tubiashii to see if there are antibiotic properties/a measurable immune response in the hemocytes. Measure by number of hemocytes in culture after 24 hours and gene expression of hemocytes.

OA & Vt class project
Prepped for larvae arrival. Assembled system with 8 chambers, filled with seawater. Hooked up air pump to provide air to each of the chambers through 1 mL serological pipettes. Larvae (C. gigas) arrived ~4:30 pm from Taylor Shellfish (shipped from Quilcene on 7.29.10). Larvae were 10 days old at shipping date and are diploids. Received ~2 million larvae (many more than currently needed but will maintain extra for future experiments and hopefully not have to order more). Poured all larvae into a 5 gallon bucket and aliquoted 150 mL of seawater + larvae into each of the chambers. Fed 25 mL of algae mix to each chamber and put a bunch of algae in bucket with larvae.

Did a gram stain of 10 uL of culture D and 10 uL of control. Nothing stained in the control and gram negative rod bacteria (V. tubiashii!) stained in culture D. The bacteria were clumped together. Indicates that the right bacteria grew and there was nothing contaminating the media.
Inoculated 40 mL of media with 100 uL of culture A and another 40 mL with 100 uL from culture D. Also made another tube of 40 mL of control. Put all tubes on shaker.

Anemone heat stress: Protein extraction Looked at gels from yesterday. Best protein concentration was 30 uL loaded in the well. Remade protein+reducing buffer mix and loaded 2 wells on new gel: one for coomassie staining and one for Western blot.

Gel 2Redid protein gel with just 30 uL of sample. Also did Western blot from gel with hsp anitbody. Protein gel looked the same as before, WB did not work. Wonder if because protein didn't transfer or had incorrect orientation of gel since did not use ladder. SR redid WB last night and had similarly failed results. Antibody probably does not work.

Oyster (O. edulis) Dissection
Dissected European flat oyster by shucking it at the hinge, twisting open a little and then scraping the adductor muscle and mantle from the top shell. Using a 3.5-gauge syringe, pierced the pericardial cavity and removed some hemolymph. Put 2 spots of hemolymph on a slide. Removed heart - ventricle and auricles - from the pericard. cavity. Cut the ventricle from the auricles, dried it slightly on a kimwipe and blotted it ~15 times on a slide. Put both slides in MeOH for a couple of minutes and then into Giemsa stain for a few minutes. The hemocytes stained purple and were very round. The heart blots had pink and purple, small round cells. No Bonamia was detected.
Notched 2 O. edulis and used syringe + needle to remove hemolymph from the pericardial cavity. Got about 12 mL of liquid from one oyster and about 3 from the other. Put a small amount of of hemolymph on a slide and plated the rest on 2 different marine agar plates. The slides were similarly stained with Giemsa stain. Slides were not stained very long but there appeared to be hemocytes in the fluid extracted from the oysters.

Anemone heat stress: Protein extraction Protocol here . Changes to protocol: 0.25 mg of tissue were used, which was an entire small anemone (control). Homogenized diced up anemone in 0.5 mL of tissue lysis buffer then added an additional 0.5 mL of buffer and mixed well before spinning down. Removed supernatant and diluted sample 1:10 in cell lysis buffer. Denatured the protein at ~90+degrees C for 5 minutes and spun down. Mixed 50 uL of denatured protein with 50 uL of 2x reducing sample buffer. Loaded Invitrogen SDS-Page gel with the protein+buffer at 3 different volumes: 10 uL, 30 uL, and 50 uL. One well of standard ladder on each gel. Run the gel at 150V for 45 minutes. Notched upper right corners of gels. Stained according to protocol in Coomassie blue and then did washes of 10% acetic acid. Did last wash overnight (changed ~2:30 am).

July 27, 2010Vt culture Plates were streaked with V. tubiashii 7.23.10. Inoculated 4 tubes of 40 mL of marine broth (2216) from different colonies on the plate. Also made one tube of 40 mL of control broth (no inoculation). Put tubes horizontally on shaker (belly dancer) at 10:30 am.

ISH: Abalone RLO Day 2 of in situ hybridization. See link in Day 1 for protocol. Changes to protocol: step 6 under "Detection" incubated overnight because no color apparent after 15 minutes.

July 26, 2010ISH: Abalone RLO Day 1 of in situ hybridization of rickettsial-like organism (RLO) parasite in abalone digestive tissue. Plates were already made (and paraffinized). Protocol for Day 1 is here . Changes to protocol: used safeclear instead of xylene, used alternate prehybridization buffer. Ended day with hybridization 53C incubation.

July 24, 2010Littorina: qPCR results Expression results in Littorina data sheet . All negative controls had evidence of contamination. ETS08 did not amplify for c-jun kinase and one sample of TY6 did not either. Expression was calculated based on the equation: arbitrary expression value = 10^(-(0.3012*Ct)+11.434). When 2 expression values were present, expression was averaged between values.

Arminia microbiologyPicked a colony from quadrant 1 on Healthy 2 plate and quad 1 on Disease 2 plate. Transferred to a slide and suspended each in a drop of nanopure H2O. Spread water + colony on slide and heat fixed/dried by passing through a flame. Once dry did gram stain: violet for 1 minute, rinse in DI H2O, iodine for 1 minute, destain briefly, counter-stained for 30 s. Rinsed thoroughly and dried. at 100x both slides had rod-shaped and coccoid gram-negative bacteria.

Littorina RNA Extractions
Extracted RNA from L. scutulata bodies from July 20. Used Tri reagent following manufacturer's protocol. Cut up tissues with a razor blade before homogenization in Tri. Final RNA was resuspended in 50 uL DEPC H2O (concentrations unknown).

Seagrass Plating
Plated pieces of diseased and healthy grasses on TCBS and marine agar. Also put strips of sterilized diseased and healthy grasses in QPX media (2 mL media in 6 well plates).

July 21, 2010Arminia microbiology etc. Arminia nudibranchs with different stages of disease (skin lesions, precursors to mortality). Arminia are from near Tacoma. Our Arminia was from the healthy tank but had evidence of disease. Swabs were streaked on 2 different plates: marine agar and TCBS. Swabs are labeled: healthy 1, healthy 2, disease 1, disease 2. Healthy 1: non-lesioned skin towards anterior, left side Healthy 2: head Disease 1: large lesion towards posterior Disease 2: right anterior lesion Swabs were done with a new cotton swab for each of the 4. Plates were divided into 4 and serial dilutions were streaked between quadrants 1-4, quad 1 being the original swab. Streakers were sterilized in between streaking with bleach & water or with EtOH. All plates were parafilmed and left to incubate at RT.

CF scraped a diseased lesion and healthy skin to make slides. There were many ciliated paramecium from the lesion (probably secondary infection) and none in the healthy skin. 4 pm did gram stain of smeared slides from skin lesions.

DNA Extraction from Littorina for microbes Extracted DNA from Littorina foot using Qiagen DNeasy Stool Kit (for Protocol see lab page, Day 2 DNA Extraction ). Samples processed were TY6 and ETS08. Both weighed less than 0.01 g. TY6 is L. scutulata, no infection and ETS08 is L. scutulata infected. Samples were eluted in 100 uL AE buffer and stored at 4C in microfuge tubes.

July 19, 2010Geoduck dissection.
Opened geoduck (Panopea generosa) by using a spatula to scrape adductor muscles off of the interior shell wall. Inside the geoduck were 2 small crabs (!), one of which had an abdomen full of eggs. After opening shell proceeded to dissect visceral ball, which was positioned off-center towards the umbo. Ctenidia were large but delicate and a light pink color (thinner with smaller/more delicate ciliated grooves than in Crassostrea gigas).
Mantle was thin and cream-colored with light brown border.
Crystalline style was very long and pretty rigid and a transparent off-white. We examined a cross-section under the microscope but it did not have much structure.
Digestive gland was full of digested brown food and some sand.
Foot was pretty large (>1") and the same color as the visceral ball/mantle.
Cut open the siphon. The outer skin was very tough and dark brown/grey. There were two channels, incurrent and excurrent siphons, divided by a muscular septum and made of very muscular tissue. On the mantle at the base of the siphon were 2 light orange small fleshy balls (LC says they appear sometimes in geoduck and can be associated with disease, but not always, are usually harmless).